Significance Nucleic acid vaccination has several advantages over conventional protein immunization. This new method has been extensively tested in mice but there have been few tests of it in larger animals. Objectives The present study was carried out to optimize nucleic acid vaccination in primates. We have varied the dose of DNA and the injection volume using intramuscular immunization and tested the dose for intradermal vaccination. Results We measured both humoral and cellular immune responses after vaccination of each animal with three different antigens. We find that immune responses appear 2-3 months after vaccination which is much slower than that seen in rodents. However, surprisingly small amounts of DNA are needed to vaccinate a macaque. We find optimal DNA doses of 200 micrograms of DNA are needed for intramuscular injection and 50 micrograms for intradermal injection. Thus only a 5 fold increase in DNA is needed when comparing primates to rodents even though they differ in body weight by 600 times. The volume of injection is also important for intramuscular injection. The immunity induced in primates is comparable to the protective levels we see in rodents. Future Directions These experiments have established conditions needed for an efficient DNA vaccination in primates. We will use this knowledge in our next experiment to vaccinate animals against SIV. KEYWORDS SIV, vaccine, nucleic acid vaccine